The long-term goal is to understand the cellular mechanisms that regulate the glycosylation of lipids and proteins in the rough endoplasmic reticulum (RER) and Golgi apparatus (GA). We propose to: (1) Continue our studies on the mechanism of glycosylation in the GA. We will attempt to identify, purify, and reconstitute into liposomes the putative sugar nucleotide translocator proteins recently characterized in the GA membrane from rat liver. Specific radiolabeled sugar nucleotides with photoreactive groups in the nucleotide moieties will be used. These studies will also be done with Golgi vesicles from wild-type and CMP-sialic acid transport negative and UDP-Galactose transport negative Chinese hamster ovary cells recently characterized in our lab. Antibodies against these translocators will be obtained and used to localize the translocators within the GA in vivo and to determine the relationship between the translocators and corresponding glycosyltransferases in the GA membrane. (2) Continue our studies on the mechanism of glycosylation in the RER. Pulse-chase experiments with rough microsomes and radioactive sugar nucleotides will be done to determine the topography of glycosylated lipid oligosaccharides in these vesicles. We shall also attempt to identify, purify, and reconstitute the UDP-Glucose and UDP-GlcNAc translocators of the RER into liposomes using approaches similar to those described for the GA. (3) Further characterize CMP-sialic acid synthetase, a nucleoplasmic enzyme. We shall complete the purification of the enzyme and prepare polyclonal and monoclonal antibodies against the synthetase. These will be used to study the site of synthesis and localization of the enzyme within the cell and in longer-term studies to attempt to understand how such proteins migrate to the nucleus. (4) Attempt to obtain subpopulations of rat liver Golgi vesicles selectively enriched in different Golgi enzyme activities. Nucleotides and antibodies against sugar nucleotide translocators that have been coupled to Sepharose will be used. If successful, these vesicles may be of use to study the transport of glycoproteins within the GA. (5) Continue to complement the above studies in vitro by attempting to isolate and characterize mutant Chinese hamster ovary cells deficient in the corresponding reactions in vivo. Cells which have been subjected to radioactive suicide with tritiated sialic acid and fucose will be screened for mutations via replica plating autoradiography, followed by biochemical and genetic complementation analyses.
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